WO2022114795A1 - Pharmaceutical composition comprising high content of active ingredient from natural product - Google Patents

Abstract

An aspect of the present disclosure relates to a formulation comprising, as an active ingredient, a high content of an herbal medicine extract from Clematis mandshurica, Trichosanthis radix, and Prunella vulgaris, which are effective in the treatment or amelioration of arthritis or the like. The formulation according to an aspect of the present disclosure is small in size regardless of the inclusion of a high content of the active ingredient, and thus has excellent convenience of medication. Even if administered twice a day, the formulation may have a release pattern and release properties that exhibit excellent therapeutic effects on arthritis. Another aspect of the present disclosure relates to a formulation comprising 280-320 mg of an herbal medicine extract from Clematis mandshurica, Trichosanthis radix, and Prunella vulgaris, wherein the formulation is administered twice a day. The formulation has a specific release pattern, and thus exhibits an excellent therapeutic effect on arthritis.

Description

Pharmaceutical composition comprising a high content of active ingredients derived from natural products

This application claims priority based on Korean Patent Application No. 10-2020-0159042 filed on November 24, 2020, and all contents disclosed in the specification and drawings of the application are incorporated herein by reference.

The present disclosure relates to a formulation containing a herbal extract of Wiryungseon, Gwalugeun and Hagocho. In particular, the present disclosure relates to a formulation that can be taken twice a day, and relates to a formulation that contains a high content and has a release pattern that exhibits excellent drug efficacy.

Osteoarthritis, also called degenerative arthritis, is the most common arthritis in adults. Osteoarthritis patients are rapidly increasing as an aging society progresses, leading to a decrease in the individual patient's quality of life as well as social and economic losses.

Arthritis refers to a disease that causes inflammation in the joints, and there are many types. The most common arthritis is osteoarthritis, and other types include rheumatoid arthritis, gout, and psoriatic arthritis. The cause of each arthritis is different, but the destruction of cartilage tissue is the same.

Degenerative arthritis (osteoarthritis) is a representative chronic joint disease that occurs most frequently among arthritis. In degenerative arthritis, the cartilage tissue surrounding the ends of the bones is gradually worn, causing inflammation and severe pain in the joints, and abnormal hardening of the bones under the cartilage. Therefore, the cartilage that cushions the friction caused by the movement of the joint is worn away and severe pain and movement disorders appear when the joint is moved.

Despite many studies to control degenerative arthritis, a fundamental treatment based on the pathological cause of the disease has not yet been developed. -Modifying osteoarthritis drug (DMOAD), etc. are mainly used for anti-inflammatory and pain relief. In addition, chondroprotectors such as hyaluronic acid, glucosamine, and chondroitin have been developed and marketed, but their therapeutic effects have not been established in cartilage protection and regeneration induction.

On the other hand, the complex herbal extracts of Wiryongseon, Gwalugeun, and Hagogi are known to have anti-inflammatory, analgesic, joint protective and immunosuppressive actions, and are widely used as therapeutic agents for osteoarthritis and rheumatoid arthritis.

These herbal extracts may be manufactured into tablets containing a high content, but if the dose is simply increased to increase the medicinal effect, an increase in side effects may appear. In addition, the currently marketed formulations containing the complex herbal extracts of Wiryungseon, Gwalugeun, and Hagocho are to be taken three times a day, so the convenience of taking is inferior.

Therefore, in order to prevent side effects caused by the high content formulation and to increase the convenience of taking, a formulation with delayed release may be considered. it turned out

Therefore, the problem to be solved by the present disclosure is that it contains a high content of Wiryungseon, Gwalugeun and Hagocho extract as active ingredients, while the formulation size is small, so it is convenient to take, and it has a release pattern that can show desirable medicinal effects, so that it is taken twice a day To provide possible formulations, in particular tablets.

In order to solve the above problems, one aspect of the present invention contains 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho as active ingredients, and the content of the active ingredient is the total weight of the mixture before coating uncoated tablets or empty capsules. It is 40 to 90% by weight (preferably, 50-90% by weight, more preferably 60-90% by weight) compared to, and containing ethyl cellulose, ethyl acrylic acid methacrylate copolymer or a mixture thereof to control the release It provides a formulation, in particular a tablet, characterized in that.

The present inventors tried to prepare a twice-daily dosage formulation containing a high content of herbal extracts of Wiryungseon, Gwalugeun and Hagocho, but unexpected problems were revealed as a result of the study. Specifically, it was possible to delay or control the release by using various binders, but in this case, it was difficult to completely elute all the extracts included. In an additional study, it was possible to achieve delayed release and good release by reducing the relative content of the extract by using a relatively large number of excipients and disintegrants. However, in this case, the size of one preparation to be taken was too large, which made it inconvenient to take. That is, due to the specific physical properties of the herbal extracts of Wiryeongseon, Gwalugeun and Hagochi, a high content of the Wiryyeongseon, Gwalugeun and Hagocho herbal extracts are included in a high weight% relative to the weight of the formulation, while having a good release pattern and high release properties at the same time It was not easy to achieve.

The present inventors have completed one aspect of the present invention by confirming that these contradictory problems can be solved by using ethyl cellulose, methacrylate ethyl acrylic acid copolymer or a mixture thereof as a binder.

As the herbal extract of Wiryyeongseon, Gwalugeun, and Hagogi of the present invention, the extract of 'Wiryeongseon·Gwalugeun·Hagocho 30% ethanol extract (40→1)', which is a yellowish-brown-brown hygroscopic powder, can be used. For example, the herbal extract of Wiryongseon, Gwalugeun and Hagocho may be a herbal extract prepared according to the method described in Korean Patent Registration Nos. 0180567 and 0483707 (WO2002-094301 A1).

Of the two binder components of the present invention, ethyl cellulose is more preferable, and therefore, a preferred embodiment of the present invention contains 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho as active ingredients, The content is 40 to 90% by weight (preferably, 50-90% by weight, more preferably 60-90% by weight) based on the total weight of the uncoated tablet before coating, and formulation characterized in that the release is controlled by including ethyl cellulose , in particular tablets.

As such ethyl cellulose, those having a viscosity of 3-22 mPa.s when measuring the viscosity at room temperature by making a 5 wt% solution with a toluene/ethanol 80:20 (volume ratio) mixed solvent may be used.

The present inventors tried to develop a formulation containing 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho, taken twice a day. Accordingly, formulations with various release patterns (rates) were manufactured and evaluated. As will be described later, if they have a specific release pattern, the therapeutic effect of arthritis (eg, osteoarthritis (degenerative joint disease), rheumatoid arthritis, etc.) is significantly superior it happened In the case of a formulation administered twice a day, it is common to control sustained release so that the active ingredient is released for a long time, similar to Example 4-4 to be described later. However, very specifically, as shown in Figs. 4 to 10, for the purpose of treating arthritis of the herbal extracts of Wiryungseon, Gwalugeun and Hagocho, the release of the active ingredient should be slightly delayed (see Fig. 4) to be taken 3 times a day. The arthritis treatment effect was increased compared to the formulation.

That is, an aspect of the present disclosure is to provide a dissolution pattern that contains 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho, and exhibits a desirable arthritis treatment effect in a formulation taken twice a day. The dissolution pattern that can achieve the excellent arthritis treatment effect of the present invention is a paddle test method at 37 ± 0.5 ° C., 50 rpm. When the dissolution test is performed under the conditions of 900 ml of water, the release of rosmarinic acid is 40 to 70 in 45 minutes. % (more preferably 45-65%, even more preferably 45-60%), at least 75% (preferably at least 80%) at 90 minutes, and at least 80% (preferably 85%) at 120 minutes above).

If such a release pattern can be achieved, additives other than the binder or release retardant mentioned above or described later may be used. That is, the scope of the present invention is not limited by the type of the specific additive described above or to be described later as long as it is a condition capable of achieving a specific emission pattern according to the present disclosure. The specific additives described above or hereinafter are merely for achieving more preferred aspects of the present disclosure.

That is, another aspect of the present invention is a formulation containing 280-320 mg of 'wiryyeongseon·gwalugeun·hagocho 30% ethanol extract (40→1)' extract as an active ingredient, and is a formulation that is taken twice a day, In the case of dissolution test under the conditions of 900ml water medium by paddle test at 37±0.5 ℃, 50rpm speed, the release of rosmarinic acid is 40-70% at 45 minutes, 75% or more at 90 minutes (more preferably preferably 40-70% at 45 minutes, 75% or more at 90 minutes, and 80% or more at 120 minutes), a formulation for the treatment of arthritis, in particular a tablet. Here, preferably, the content of the 'wiryyeongseon·gwalugeun·hagocho 30% ethanol extract (40→1)' extract is 40 to 90% by weight relative to the total weight of the mixture before filling uncoated tablets or empty capsules (preferably preferably, 50-90% by weight, more preferably 60-90% by weight).

In a preferred embodiment of the present invention, the preparation for treating arthritis is a tablet or capsule preparation containing 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho as active ingredients, and is a preparation to be taken twice a day, 37± In the case of dissolution test under the conditions of 900 ml of water medium with a paddle test method at 0.5 ° C., 50 rpm, the release of rosmarinic acid is 45-65% at 45 minutes, 80% or more at 90 minutes, 85% or more at 120 minutes (more Preferably, 45-60% at 45 minutes, 80% or more at 90 minutes, 85% or more at 120 minutes). Here, preferably, the content of the 'wiryyeongseon·gwalugeun·hagocho 30% ethanol extract (40→1)' extract is 40 to 90 weight compared to the total weight of the capsule filling excluding the weight of the uncoated tablet or empty capsule before coating % (preferably 50-90% by weight, more preferably 60-90% by weight).

Preferably, a preferred aspect of the present invention is a tablet containing 280-320 mg of 'Wiryyeongseon·Gwalugeun·Hagocho 30% ethanol extract (40→1)' extract as an active ingredient, and the content of the active ingredient is 40 to 90% by weight (preferably, 50-90% by weight, more preferably 60-90% by weight) relative to the total weight, it is a tablet to be taken twice a day, 37±0.5° C., a paddle at a speed of 50 rpm ( In the case of dissolution test under the conditions of 900 ml of water as a paddle test method, the release of rosmarinic acid is 40-70% at 45 minutes, 75% or more at 90 minutes (more preferably 40-70% at 45 minutes, 75 at 90 minutes) % or more, 80% or more at 120 minutes, even more preferably 45-65% at 45 minutes, 80% or more at 90 minutes, and 85% or more at 120 minutes), wherein the controlled release is ethyl cellulose, methacryl Provides a tablet comprising an acid ethyl acrylic acid copolymer or a mixture thereof (more preferably, ethyl cellulose).

One aspect of the present invention is a tablet containing 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho as active ingredients, and the content of the active ingredient is 40 to 90% by weight (preferably, 50-90% by weight, more preferably 60-90% by weight), and it is a tablet taken twice a day, and dissolution test under the conditions of 900ml water medium by paddle test at 37±0.5°C and 50rpm speed hour, the release of rosmarinic acid is 40-70% at 45 minutes, 75% or more at 90 minutes, 80% or more at 120 minutes (more preferably 45-65% at 45 minutes, 80% or more at 90 minutes, 120 minutes to 85% or more), and provides a tablet characterized in that the release is adjusted using ethyl cellulose. The content of the ethyl cellulose is preferably 0.1-10% by weight, more preferably 0.4-5% by weight, even more preferably 0.8-3% by weight, based on the total weight of the uncoated tablet before coating.

In one aspect of the present invention, the tablet may optionally further include a pharmaceutically acceptable excipient, adsorbent, disintegrant, lubricant, and the like.

In one aspect of the present invention, various materials such as polyvinylpyrrolidone, ethyl cellulose, methacrylate ethyl acrylic acid copolymer, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and acrylic acid copolymer may be used as the release controlling agent. have. However, in terms of reducing the size of the tablet and achieving good release properties, ethyl cellulose, ethyl methacrylate copolymer, or a mixture thereof is preferable. The controlled-release material is preferably 0.1-10 wt%, more preferably 0.4-5 wt%, even more preferably 0.8-3 wt%, based on the total weight of the uncoated tablet before coating.

Microcrystalline cellulose, lactose, mannitol, etc. can be used as the excipient, and the content of these excipients is preferably 5-40 wt%, more preferably 10-35 wt%, and 15-30 wt%, based on the weight of the uncoated tablet before coating. is even more preferable.

As the adsorbent, light anhydrous silicic acid (including hydrophobic light anhydrous silicic acid), magnesium aluminum silicate, etc. can be used, and the content of this adsorbent is preferably 0.5-9 wt%, more preferably 1-7 wt%, based on the weight of the uncoated tablet before coating. preferred, 2-5% by weight is even more preferred.

As the disintegrant, croscarmellose sodium, sodium starch glycolate, crospovidone, low-substituted hydroxypropyl cellulose, etc. may be used. However, croscarmellose sodium, low-substituted hydroxypropyl cellulose, or a mixture thereof is preferable in terms of reducing the size of the tablet and achieving good release properties. When croscarmellose sodium, low-substituted hydroxypropyl cellulose, or a mixture thereof is used, good release property is achieved even under conditions where the content of the active ingredient is increased. Among croscarmellose sodium or low-substituted hydroxypropyl cellulose as the disintegrant, croscarmellose sodium is particularly preferred for the purpose of the present invention. The content of this disintegrant is preferably 1-10% by weight, more preferably 1-8% by weight, even more preferably 2-5% by weight, based on the weight of the uncoated tablet before coating.

As the lubricant, stearyl fumarate, magnesium stearate, stearic acid, talc, etc. can be used, and the content of the lubricant is preferably 0.1-4 wt%, more preferably 0.2-2 wt%, based on the weight of the uncoated tablet before coating. do.

In one aspect of the present invention, the tablet according to the present invention may also be coated for various purposes, such as protection from external impact, appearance, control of release, and the like. For example, the uncoated tablet may be coated using a coating composition including a film former, a plasticizer, an anti-adhesive body, and the like, and the content of the coating is 1-15 wt%, preferably based on the total weight of the uncoated tablet prior to coating. can be coated in an amount of 3-10% by weight. As the film coating composition (product), various coating-based products including polyvinyl alcohol sold by ColorCon as a basic coating material may be used.

A preferred aspect of the present invention is a tablet containing 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho as active ingredients, and the content of the active ingredient is 40 to 90% by weight (preferably compared to the total weight of the uncoated tablet before coating) , 50-90% by weight, more preferably 60-90% by weight), and is a tablet to be taken twice a day, and eluted under the conditions of 900ml of water by a paddle test at 37±0.5° C. and 50rpm speed. In the test, the release of rosmarinic acid is 40-70% at 45 minutes, 75% or more at 90 minutes, 80% or more at 120 minutes (more preferably 45-65% at 45 minutes, 80% or more at 90 minutes, 120 85% or more per minute), controlled release using ethyl cellulose, and provides a tablet characterized in that it contains croscarmellose sodium as a disintegrant.

The formulation according to the present invention is obtained by preparing granules by a wet granulation method using a solvent such as water, ethanol, isopropanol, or a dry granulation method using compression force, and then mixing the granules with post-mixing components such as a disintegrant and a lubricant. It may be prepared as a tablet or capsule. The formulation according to the present invention may be manufactured into tablets by a direct tapping method without a granule manufacturing process. However, the wet granulation method may be preferable in terms of maintaining the release pattern without deviation for achieving one of the various objects of the present invention.

Therefore, one aspect of the present invention is to prepare granules using a herbal extract of Wiryungseon, Gwalugeun and Hagocho, an adsorbent, a binder, optionally an excipient, optionally a disintegrant; preparing a tablet by post-mixing a lubricant, a disintegrant, etc., which are pharmaceutically acceptable additives, to the granules prepared above and tableting; and optionally coating the tablet.

One aspect of the present disclosure provides a formulation containing a high content of herbal extracts of Wiryyeongseon, Gwalugeun and Hagocho, and can be taken twice a day. The formulation according to the present disclosure has a specific release pattern and exhibits excellent arthritis treatment effect even when taken twice a day. The formulation of one aspect according to the present disclosure also contains a high content of herbal extracts of Wiryungseon, Gwalugeun and Hagocho, and thus has a small size and excellent convenience in taking it.

Since the following drawings attached to the present specification serve to further understand the technical idea of the present invention together with the contents of the above-described invention, the present invention should not be construed as limited only to the matters described in such drawings.

1 is a graph showing the dissolution pattern in a pH 1.2 medium for each type of binder.

2 is a graph showing the dissolution pattern in a pH 6.8 medium for each viscosity of ethyl cellulose.

3 is a graph showing the dissolution pattern in water of Examples and Comparative Examples according to the present invention.

4 is a graph showing the dissolution patterns in water of formulations having various dissolution patterns.

5 is a result of evaluating the effect of formulations with various dissolution patterns on gait after Meniscectomy and anterior cruciate ligament resection.

6 is a result of evaluating the effect of formulations having various dissolution patterns on arthritis, a weight bearing test (Incapacitance test) results.

7 is an ELISA analysis result evaluating the effect of agents having various dissolution patterns on cytokines in arthritis.

8 is an area histochemical staining analysis result evaluating the effect of formulations with various dissolution patterns on biomarkers in arthritis.

9 is a result of evaluating the effects of agents having various dissolution patterns as OARSI scores by histopathological analysis.

10 is an immunohistochemical analysis result, showing the results of Hematoxylin & Eosin (H&E) and Safranin-O staining of the joint surface, cartilage and synovium after completion of the test.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art to which the present invention pertains.

Example 1: Confirmation of dissolution patterns using various binders

Using the contents and ingredients shown in Table 1 below, tablets containing 30% ethanol extract of Wiryungseon·Gwalugeun·Hagocho 30% (40→1) extract were prepared. The extract, crospovidone, hydrophobic light silicic anhydride, and binder were mixed. Then, magnesium stearate was added, mixed, and tableted.

Ingredients (unit: mg)	Example 1-1	Example 1-2	Example 1-3	Example 1-4	Example 1-5	Example 1-6
herbal extract	300	300	300	300	300	300
Hydrophobic light anhydrous silicic acid	15	15	15	15	15	15
polyvinylpyrrolidone	10.5
ethyl cellulose		10.5
Ethyl methacrylate copolymer			10.5
Hydroxypropylmethylcellulose					10.5
Hydroxypropyl Cellulose				10.5
acrylic acid copolymer						10.5
crospovidone	20	20	20	20	20	20
magnesium stearate	2.5	2.5	2.5	2.5	2.5	2.5
tablet total weight	348	348	348	348	348	348

Polyvinylpyrrolidone: Kollidon K30, viscosity about 5.5-8.5 cps (20℃, 10% w/v)

Ethyl methacrylate copolymer: Eudragit L100-55, viscosity about 50~200cps

Hydroxypropylmethylcellulose: Metolose, viscosity about 100cps (20℃/2% w/v)

Hydroxypropyl cellulose: Nisso HPC-L, viscosity about 6~10cps (20℃/2% w/v)

Acrylic acid copolymer: Carbomer 971, viscosity about 4000~11000 (0.5%w/w)

Experimental Example 1: Evaluation of dissolution by type of binder

The dissolution test of the tablets prepared in Example 1 was performed according to the dissolution test method of the 12th revision of the Korean Pharmacopoeia. The paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0 ± 0.5 °C. The eluate was carried out at a pH of 1.2 900 ml. In order to improve the stability of the sample solution, it was analyzed after pretreatment with acetonitrile. The dissolution test results are shown in Table 2 and FIG. 1 below. The dissolution rate was measured by measuring the content of rosmarinic acid, an active ingredient of the herbal extract, over time. The content of rosmarinic acid is described in J. Sep. Sci. 2015, 0, 1-8 (“Quality evaluation of Salvia miltiorrhiza Bge. by ultra high performance liquid chromatography with photodiode array detection and chemical fingerprinting coupled with chemometric analysis”).

hours (minutes)	Example 1-1	Example 1-2	Example 1-3	Example 1-4	Example 1-5	Example 1-6
5	3	22	21	5	10	4
10	10	38	38	8	23	8
15	19	47	45	10	34	12
30	37	62	59	18	62	30
45	47	73	70	27	77	47
60	52	80	77	37	83	63
90	60	92	86	56	84	72
120	66	97	94	66	85	76
180	76	100	98	76	85	80
240	81	101	101	80	85	82

As shown in Table 2, Examples 1-1, 1-4, and 1-6 showed a large dissolution delay effect, and Examples 1-2 and 1-3 showed a similar dissolution delay effect. In Examples 1-5, the dissolution delay effect was small. However, in the case of the remaining examples except Examples 1-2 and 1-3, it was confirmed that the dissolution amount did not reach about 80% even when the release was evaluated for a very sufficient time. It was found to be practically impossible to use. This is presumed to be due to the combination of the properties of the specific extract used in the present invention and the properties of the polymer, for example, it is thought to be related to the function of preventing the gelation of the herbal extract, etc., but the present invention is It is not limited to these theoretical mechanisms.

The ethyl methacrylate copolymer used in Examples 1-3 showed a desirable release pattern in an acidic medium, but there was a problem in that the release pattern was different in other pH media, making it difficult to control the release, and as a result, Example 1 Less preferred than -2 ethyl cellulose.

The present inventors also performed various evaluations and judged that the dissolution pattern as in Examples 1-2 or 1-3 in a pH 1.2 medium is preferable as the dissolution pattern of the formulation containing 300 mg tablets twice taken, so in this aspect There is also the problem that binders other than Examples 1-2 and 1-3 delay the release too much.

As a result, surprisingly, ethyl cellulose and methacrylate ethyl acrylic acid copolymer are preferable than commonly used HPMC, etc. , ethyl cellulose was more preferable.

Example 2: Confirmation of delayed dissolution pattern using ethyl cellulose

Using the contents and ingredients shown in Table 3 below, tablets containing 30% ethanol extract of Wiryungseon·Gwalugeun·Hagocho 30% (40→1) extract were prepared. First, ethyl cellulose was dissolved in isopropyl alcohol to prepare a binding solution, and then the extract and hydrophobic light silicic anhydride were added to the binding solution to prepare wet granules. Crospovidone and magnesium stearate were mixed with the prepared wet granules and tableted.

Ingredients (unit: mg)	Example 2-1	Example 2-2	Example 2-3	Example 2-4
herbal extract	300	300	300	300
Hydrophobic light anhydrous silicic acid	15	15	15	15
Ethyl Cellulose 4cps	20
Ethyl Cellulose 7cps		20
Ethyl Cellulose 10cps			20
Ethyl Cellulose 20cps				20
crospovidone	15	15	15	15
magnesium stearate	2	2	2	2
tablet total weight	352	352	352	352

Experimental Example 2: Evaluation of dissolution by viscosity of ethyl cellulose in pH 6.8 medium

The dissolution of Examples 2-1 to 2-4 was evaluated according to the dissolution test method of the 12th revision of the Korean Pharmacopoeia. The paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0 ± 0.5 °C. The eluate was carried out at pH 6.8 900ml. In order to improve the stability of the sample solution, it was analyzed after pretreatment with acetonitrile. The dissolution test results of rosmarinic acid are shown in Table 4 and FIG. 2 below.

hours (minutes)	Example 2-1	Example 2-2	Example 2-3	Example 2-4
5	12	10	9	6
10	13	12	14	11
15	17	17	17	16
30	27	25	25	25
45	34	33	32	33
60	43	39	39	39
90	65	60	52	56
120	92	85	72	78
180	96	95	94	92
240	96	95	95	93

As shown in Table 4, the formulation of the present invention using ethyl cellulose exhibited a good release pattern even at pH 6.8, and could exhibit a release close to 100% even at pH 6.8. In addition, from Example 2-1 to 2-4, the dissolution rate is a slower pattern, which is consistent with the increase in the viscosity of ethyl cellulose.

Example 3 and Comparative Example 1: Evaluation of dissolution according to various components

Tablets were prepared according to the contents and ingredients shown in Table 5 below. Example 3 was prepared by wet granulation of herbal extracts, hydrophobic light anhydrous silicic acid, and ethyl cellulose of Wiryungseon, Gwalugeun and Hagocho. Microcrystalline cellulose, croscarmellose sodium and stearyl fumarate were mixed in the prepared wet granules and tableted. The core thus prepared was coated with a coating base. Comparative Example 1 was prepared by dry mixing of herbal extracts of Wiryungseon, Gwalugeun and Hagocho, light anhydrous silicic acid, corn starch, microcrystalline cellulose and sodium starch glycolate. Magnesium stearate was post-mixed with the prepared mixture, followed by tableting. The core thus prepared was coated with a coating base.

Ingredients (unit: mg)	Example 3	Comparative Example 1
herbal extract	300	200
Microcrystalline Cellulose	95	113
corn starch		50
Light anhydrous silicic acid		10
Hydrophobic light anhydrous silicic acid	10
Ethyl Cellulose 4cps	8
Croscarmellose Sodium	13
Sodium starch glycolate		25
magnesium stearate		2
Stearyl Fumarate	2
coating base	28	30
tablet total weight	456	430

Experimental Example 3: Evaluation of dissolution of Example 3 and Comparative Example 1

The dissolution patterns of Example 3, Comparative Example 1 and Comparative Example 2 were evaluated according to the 12th revision dissolution test method of the Korean Pharmacopoeia. The paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0 ± 0.5 °C. The eluate was carried out in 900 ml of purified water. In order to improve the stability of the sample solution, it was analyzed after pretreatment with acetonitrile and pH 1.2 buffer. The dissolution test results of rosmarinic acid are shown in Table 6 and FIG. 3 below.

hours (minutes)	Example 3	Comparative Example 1
5	5	6
10	14	24
15	20	39
30	40	67
45	57	81
60	71	82
90	88	84
120	94	85
180	98	86
240	99	86

As shown in Table 6, the formulation according to the present invention exhibited a good release pattern even in water. The present inventors performed various evaluations and when the dissolution medium is water, the dissolution pattern as in Example 3 (for example, 40 to 70% at 45 minutes, 75% or more at 90 minutes, more preferably 85% at 120 minutes % or more) was found to be desirable, but it was not easy to achieve the dissolution pattern as described above. For example, the dissolution result of Comparative Example 1 shows that the final dissolution rate is not high even though the initial dissolution rate is increased by using a lot of excipients and disintegrants and lowering the content of the extract.

Example 4: Evaluation of drug efficacy according to dissolution pattern

The effect of the dissolution pattern on the drug efficacy was evaluated after preparing Wiryungseon·Gwalugeun·Hagocho 30% Ethanol Extract (40→1) tablets with various dissolution patterns with the prescription shown in Table 7 below. 200 mg immediate-release tablet, 300 mg immediate-release tablet, 300 mg delayed-release tablet, and 300 mg sustained-release tablet were prepared. First, a binder was prepared by dissolving a binder in a suitable solvent among water, ethanol, isopropyl alcohol, or a mixed solvent thereof. Then, the extract, light anhydrous silicic acid, excipients, etc. were added to the binding solution to prepare wet granules. A disintegrant and a lubricant were mixed with the prepared wet granules and tableted. Thereafter, film coating was performed in a conventional manner.

Ingredients (unit: mg)	Example 4-1	Example 4-2	Example 4-3	Example 4-4
	200mg immediate release tablet	300mg immediate release tablet	300mg delayed release tablet	300mg sustained release tablet
Gwalugeun, Wiryongseon, Hagocho 30% Ethanol Extract (40:1)	200	300	300	300
Light anhydrous silicic acid (glidant)	10	15	20	20
Corn Starch (Binder)	50
Povidone K30 (binder)		5
Ethyl Cellulose (Binder)			4	4
Microcrystalline Cellulose PH102 (Excipient)	113	85	115	115
Kollidon SR (sustained release agent)				50
Sodium starch glycolate (disintegrant)	25
Crospovidone (disintegrant)		15
Croscarmellose sodium (disintegrant)		25	10	10
Magnesium stearate (glidant)	2	5
Sodium stearyl fumarate (glidant)			5	5
film coating	30	27	29	30
tablet total weight	430	477	483	534

Experimental Example 4-1: dissolution evaluation

Dissolution of 200 mg immediate-release tablet, 300 mg immediate-release tablet, 300 mg delayed-release tablet, and 300 mg sustained-release tablet was evaluated in the same manner as in Experimental Example 3. That is, the paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0±0.5°C. The eluate was carried out in 900 ml of purified water. The dissolution results of rosmarinic acid are summarized in Table 8 and FIG. 4 below.

time/dissolution % (n=6)	Example 4-1	Example 4-2	Example 4-3	Example 4-4
0	0	0	0	0
5	3	6	3	2
10	24	21	9	5
15	45	36	15	7
30	85	71	31	12
45	98	93	49	16
60	100	102	66	20
90	102	106	88	29
120	104	106	95	38
180	105	105	100	57
240	106	106	103	71
360	106	105	105	89

When the average dissolution rate reached 80%, it was confirmed that '200mg tablet immediate-release' 30 minutes, '300mg immediate-release tablet' 45 minutes, '300mg delayed-release tablet' 90 minutes, and '300mg sustained-release tablet' 360 minutes. Therefore, it is judged that each dosage form has the characteristics of an immediate-release tablet, a delayed-release tablet, and a sustained-release tablet, respectively.

Experimental Example 4-2: Evaluation of osteoarthritis treatment effect

Experimental animals, test substances and test groups

The treatment effect of osteoarthritis was evaluated using the beagle dog meniscectomy and anterior cruciate ligament resection (ACLT) models. As test substances, 200 mg immediate-release tablet, 300 mg immediate-release tablet, 300 mg delayed-release tablet, and 300 mg sustained-release tablet prepared in Example 4 were used. As a positive control, Pfizer's Celecoxib formulation (manufacture (lot) number: EF6274, capsule) was used. As a negative control group (Vehicle), tablets made of the prescriptions shown in Table 9 below were used.

Ingredients (unit: mg)	Vehicle (placebo)
Microcrystalline Cellulose PH102 (Excipient)	319
Corn Starch (Excipient)	47
Sodium starch glycolate (disintegrant)	9
Magnesium stearate (glidant)	5
film coating	29
tablet total weight	409

The composition of the test group was shown in Table 10 below.

army	gender	number of animals (number of animals)	animal number	Meniscectomy	substance to be administered	dosing frequency	Dosage (mg)	Dosage (affection)
G1	M	4	1-4	N
G2	M	4	5-8	Y	Vehicle	b.i.d.	N/A	1T
G3	M	4	9-12	Y	Celecoxib	q.d.	200	1T
G4	M	4	13-16	Y	200mg immediate release tablet	t.i.d.	200	1T
G5	M	4	17-20	Y	300mg immediate release tablet	b.i.d.	300	1T
G6	M	4	21-24	Y	300mg delayed release tablet	b.i.d.	300	1T
G7	M	4	25-28	Y	300mg sustained release tablet	b.i.d.	300	1T

It was orally administered to Beagle dog (Xi'an Dilepu Biology & Medicine Co., Ltd) according to the number of administrations in Table 9, and was administered for 8 weeks. Specifically, the animals were placed in a cage with their mouths open, put the test substance inside the tongue, closed their mouths, and then swallowed by gently stroking the throat. After confirming that it was swallowed, about 10 mL of water was administered using a syringe.

Meniscectomy and Anterior Cruciate Ligament Resection

Before the operation, hair removal was performed on both knees of the animal using a clipper. The animal was anesthetized, and the area to be incised was widely disinfected using Povidone and 70% alcohol. Then, the skin of the right knee was incised. By blunt dissection of the surrounding tissue, the articular surface of the femoral distal end was exposed. A defect window was made on the medial articular surface and the anterior cruciate ligament was excised. Wound closure was performed using 4-0 nylon. In the case of the left knee, only anterior cruciate ligament resection was performed.

One week after the meniscectomy, artificial exercise (30 minutes/day) was performed on the animals once/day for 7 days.

Observation and inspection items

(1) Gait evaluation

A gait evaluation was performed to evaluate the efficacy of the formulations prepared in Example 4 on an animal model of osteoarthritis. It was conducted twice/week before and after administration of the test substance. Gait evaluation was performed according to the evaluation criteria of Table 11 below, and images were taken using a digital camera.

Score		Parameters
0	No observable lameness
One	Intermittent, mild weight-bearing lameness with little, if any, change in gait
2	Moderate weight-bearing lameness—obvious lameness with noticeable gait change
3	Severe weight-bearing lameness - "toe-touching" only
4	Non-weight-bearing

The results are shown in FIG. 5 . As shown in FIG. 5 , the results of the 300 mg delayed-release tablet (G6) were the most favorable. In particular, compared to the 300 mg immediate-release tablet (G5) and the 300 mg sustained-release tablet (G7), only the 300 mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

(2) Incapacitance test

The effect of the formulations prepared in Example 4 on weight bearing in an animal model of osteoarthritis was confirmed. The hindlimb weight bearing was measured using a foot weight tester (Incapacitance tester (1029-S, Linton instrumentation, USA). In this test, osteoarthritis-induced Beagle dog had anterior cruciate ligament resection compared to the left hindlimb where only anterior cruciate ligament resection was performed. And since the right hind limb was more severe pain when the medial meniscus was resected at the same time, I had to stand on the left hind limb, and accordingly, the weight load of the right hind limb was measured relatively lightly compared to the left hind limb weight. The weight (g) of both feet was measured in a state where the dog's stomach did not touch the device sensor, and the weight load ratio (%) was calculated by the method of Equation 1 below using the measured weight of the feet. With the support and pressing force, the weight of both feet is balanced in normal cases, so that the weight-bearing rate (%) of one foot appears at 50%.

[Formula 1]

Weight bearing rate (%) = [weight of osteoarthritis-induced hind limb / (weight of hind limb of both feet)] × 100

Before and after the administration of the test substance, the level of the weight-bearing distribution ratio of the hind limbs was measured using an incapacitance tester once/week for 8 weeks. The results are shown in FIG. 6 .

As shown in FIG. 6 , the results of the 300 mg delayed-release tablet (G6) were the most favorable. In particular, compared to the 300 mg immediate-release tablet (G5) and the 300 mg sustained-release tablet (G7), only the 300 mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

(3) ELISA analysis

In order to confirm the activity of the biomarkers related to the occurrence of arthritis, the three types of cytokines IL-1β, MMP-3 and TNF-α were analyzed as biomarkers in the joint fluid using the joint fluid collected on the day of autopsy. For the analysis, a commercially available ELISA kit was used. The results are shown in FIG. 7 .

One of the potent mechanisms inducing osteoarthritis is increased production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and collagenase and stromelysin. It is said that the secretion of MMPs such as the back is increased, causing damage to articular cartilage. That is, the expression of MMP-3, MMP-9, and MMP-13 increases when osteoarthritis occurs, and it is known that the increase in MMPs damages the collagen matrix constituting the cartilage, thereby exacerbating degenerative arthritis. have. In addition, it has been established that ADAMTS5 and MMP13 play a decisive role in the progression of osteoarthritis in the degradation of aggrecan and type II collagen, which are representative chondrocyte extracellular matrix.

There are several causes of arthritis progression. Among them, an important cause is related to the activity of macrophages (macrophages). When macrophage existing in synovial fluid is activated by PAMPs, DAMPs and inflammasome, it is polarized to M1 macrophage, which plays a role related to inflammation. This M1-macrophage secretes inflammatory cytokines (eg, IL-1B, IL-6, TNF-α), growth factors, MMPs (ex: MMP1, MMP2, MMP3, MMP13) and TIMP, as a result Inflammation. Cartilage degradation and osteophyte formation are caused. These cytokines, MMPs, and secreted proteins maintain the activity of macrophage in the form of autocrine in the form of M1, and M1-macrophage in the form of TGF-β, JNK, Akt, NF-κB and beta-catenin It promotes degradation of extracellular matrix (ECM) components by altering signaling pathways. The decomposed ECM acts as a DAMP and stimulates macrophage activation and polarization to M1-macrophage again, resulting in repeated inflammation and cartilage degradation.

As a result of confirming the levels in the joint fluid of the major cytokines (IL-1β, TNF-α) and MMPs (MMP3) secreted by macrophages that cause such arthritis through ELISA, G3, G4, G5 compared to the induced group (G2) , the level of IL-1β was significantly decreased in G6 and G7, and the level of TNF-α was significantly decreased in the test groups G3, G4, G5 and G6 compared to the induction group (G2). The level of MMP3 in the joint fluid was significantly decreased in the test group G3, G4, G5, G6 and G7 compared to the induction group (G2). As shown in FIG. 7 , the results of the 300 mg delayed-release tablet (G6) were the most favorable. In particular, compared to the 300 mg immediate-release tablet (G5) and the 300 mg sustained-release tablet (G7), only the 300 mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result. Through these results, it is thought that the level of inflammatory cytokine and MMP3 decreased, which helped relieve arthritis in the ELISA test.

(4) Immunohistochemical staining

In addition, to confirm the MMP-13 inhibitory effect and the collagen type II generation effect as a biomarker related to the development of arthritis, collagen type II and MMP-13 were analyzed as immunohistochemical staining (IHC) markers.

After the joint fluid was collected on the day of autopsy, the animal was euthanized, and the defect was visually observed and photographed. The defect was removed and fixed in 10% neutral buffered formalin solution. The collected joint fluid was stored in a cryogenic freezer set at -70 ° C or lower until analysis. The fixed tissue was subjected to general tissue processing such as demineralization, trimming, dehydration, paraffin embedding, and sectioning to prepare a specimen for histopathological examination. Then, Hematoxylin & Eosin (H&E), Safranin-O, and immunohistochemical staining (Collagen type II and MMP-13) were performed and staining was performed, and histopathological changes were performed using an optical microscope (Olympus BX53, Japan). is observed, and immunohistochemical staining is analyzed using an Image analyzer (Zen 2.3 blue edition, Carl Zeiss, Germany).

For immunohistochemical staining, MMP13 (R&D System, MAB511-500) and Collagen II (abcan, ab34712) are used as primary antibodies, and for MMP13, DAKO, Envision+System-HRP labeled polymer anti-mouse (K4001) ), Collagen II, DAKO, Envision+System-HRP labeled polymer anti-rabbit (K4003) was used as a secondary antibody. After staining with DAB, counterstaining was performed with hematoxylin.

The results are shown in FIG. 8 .

As shown in Figure 8, 300mg delayed-release tablet (G6) showed good results. In particular, compared to the 300 mg immediate-release tablet (G5) and the 300 mg sustained-release tablet (G7), only the 300 mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

(5) histopathology assay

After H&E staining, cartilage structure and chondrocyte levels were confirmed in the joint surface among OARSI score items, and synovial lining, inflammatory cell infiltration, and synovial hyperplasia levels were confirmed in the joint cavity. After Safranin-O staining, the level of proteoglycan was confirmed in the joint surface among the OARSI score items. The corresponding evaluation method was scored by referring to The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the dog (Osteoarthritis Cartilage. 2010 Oct; 18 Suppl 3:S66-79.).

The results are shown in FIGS. 9 and 10 . As shown in Figure 9, 300mg delayed-release tablet (G6) showed good results. In particular, compared to the 300 mg immediate-release tablet (G5) and the 300 mg sustained-release tablet (G7), only the 300 mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

In addition, as shown in FIG. 10 , as a result of Safranin-O staining, in the osteoarthritis-induced group, normal cartilage tissue was destroyed by induction, and the level of damage to proteoglycan tissue increased. In contrast, in the drug-administered group, a large amount of stained proteoglycan tissue was distributed around the synovial membrane. In H&E staining, the joint surface cracks and erosion were deeper in the osteoarthritis-induced group compared to the cartilage tissue in the normal group, and it was confirmed that the level of damage to the joint surface was reduced in the drug-administered group. Compared to the joint synovial tissue of the normal group, the osteoarthritis-induced group increased the level of villi due to increased damage due to excessive infiltration of synovial cells around the joint. Also, 300 mg delayed-release tablet (G6) showed good results. In particular, compared to the 300 mg immediate-release tablet (G5) and the 300 mg sustained-release tablet (G7), only the 300 mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

Claims (10)

1. It is a tablet or capsule preparation containing 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho as active ingredients, and is a preparation to be taken twice a day,

   In the case of dissolution test under the conditions of 900 ml of water by the paddle test method at 37±0.5 ℃, 50 rpm, the release of rosmarinic acid is 40-70% at 45 minutes, 75% or more at 90 minutes, 80% or more at 120 minutes Preparations for the treatment of arthritis.
2. The formulation according to claim 1, wherein the active ingredients of the herbal extracts of Wiryungseon, Gwalugeun and Hagocho are 40 to 90% by weight based on the total weight of the capsule filling excluding the weight of uncoated tablets or empty capsules before coating.
3. The formulation of claim 1, wherein the formulation is a tablet.
4. The method according to any one of claims 1 to 3, wherein the formulation is 37 ± 0.5 ° C, 50 rpm paddle test method, when the dissolution test under the conditions of 900 ml of water medium, the release of rosmarinic acid 45 in 45 minutes ~65%, 80% or more at 90 minutes, 85% or more at 120 minutes formulations.
5. The formulation according to any one of claims 1 to 3, wherein the formulation comprises ethyl cellulose, an ethyl methacrylate copolymer, or a mixture thereof to control the release.
6. 6. The formulation of claim 5, wherein the formulation comprises ethyl cellulose to control release.
7. As an active ingredient, it contains 280-320 mg of herbal extracts of Wiryungseon, Gwalugeun and Hagocho, and the content of the active ingredient is 40 to 90% by weight relative to the total weight of the mixture before coating uncoated tablets or empty capsules, ethyl cellulose, methacryl A formulation comprising an acid ethyl acrylic acid copolymer or a mixture thereof to control the release.
8. 8. The formulation of claim 7, wherein the formulation comprises ethylcellulose.
9. The tablet according to claim 7 or 8, wherein the content of the active ingredient in the formulation is 50 to 90% by weight relative to the total weight of the mixture before filling the uncoated tablet or empty capsule before coating.
10. 9. The formulation of claim 7 or 8, wherein the formulation is a tablet.

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